Effect of recombinant protein expression on baculovirus budded virus structure and infectivity

杆状病毒表达载体（BEVS）已成为生产重组蛋白最通用和最成功的系统之一。通常，它们能在其他系统失败的地方成功地制造出难以实现的目标。它们越来越多地被用于生产重组蛋白亚基疫苗，例如流感、Covid-19和几种动物疾病疫苗。它们对于生产用于药物发现的大而复杂的跨膜蛋白也非常有用。杆状病毒在昆虫细胞中的自然复制周期包括基因表达的早期、晚期和极晚期。值得注意的是，后期看到从质膜萌发的感染性病毒颗粒的产生，这对病毒在细胞培养中的传播至关重要。重组蛋白大部分在极晚阶段产生，然而，一些蛋白质合成在后期开始，如果目标是细胞毒性的，这可能会影响出芽病毒（BV）的产量。这可使病毒的感染滴度降低10倍。虽然这在监测重组蛋白的小规模测试中通常不是问题，但在随后的大规模工作中，使用高颗粒细胞比感染BEVS的大型昆虫细胞培养物可能会产生严重后果。另一个问题是生产高滴度的BV库存，在储存时可能不稳定。而不是保持高滴度数月，这段时间减少到几周的储存在一个寒冷的房间。除了与重组蛋白生产有关外，病毒低滴度和不稳定性的原因尚不清楚。我们假设BV的结构完整性受到重组蛋白的影响。然而，由于先进设施的限制，BV结构数据非常稀缺，导致缺乏对这些表达载体的深入了解，并影响了BEVS的进一步发展。利用STFC RAL和ISIS的世界领先的结构表征设施，我们可以检查和研究感染滴度或储存稳定性受损的重组BV的结构完整性。这项结构研究的成功完成将使我们能够制定重组蛋白生产的智能设计策略，以减轻它们对细菌性病毒结构和传染性的影响。这将使目前在可行性阶段失败的项目能够扩大规模，并为许多不同的学术和商业用途生产蛋白质。

Baculovirus expression vectors (BEVS) have become one of the most versatile and successful systems for the production of recombinant proteins. Frequently, they succeed in producing difficult targets where other systems have failed. Increasingly, they are employed to produce recombinant protein subunit vaccines, e.g. influenza, Covid-19 and several for animal diseases. They are also very useful for producing large, complex transmembrane proteins for drug discovery.The natural replication cycle of baculoviruses in insect cells involves early, late and very late phases of gene expression. Notably, the late phase sees the production of infectious virus particles that bud from the plasma membrane and are essential for virus dissemination in cell culture. Recombinant proteins are largely produced in the very late phase, however, some protein synthesis initiates in the late phase and if the target is cytotoxic this can have an impact on budded virus (BV) yield. This may reduce the infectious titre of virus by up to 10-fold. While this is not usually a problem in small scale tests to monitor recombinant proteins, it can have serious consequences in subsequent scale up work where large cultures of insect cells are infected with BEVS using high particle:cell ratios. A further issue is the production of BV stocks with high titres that may not be stable upon storage. Rather than maintaining a high titre for months, this period reduces to a few weeks of storage in a cold room. The cause(s) of the virus low titre and instability are unknown, apart from the link with recombinant protein production. We hypothesise that the structural integrity of BV is affected by the recombinant protein. However, BV structural data is very scarce due to limited access to state-of-the-art facilities, leading to a lack of in-depth understanding of these expression vectors and impacting on further BEVS development. With world-leading structural-characterisation facilities at STFC RAL and ISIS we can examine and investigate the structural integrity of recombinant BV with compromised infectious titre or stability on storage. The successful completion of this structural study will allow us to formulate intelligent design strategies for recombinant protein production that mitigate their effect on BV structure and infectivity. Thiswill allow projects that currently fail at the feasibilioty stage to progress to scale up and protein production for many different academic and commercial uses.